Biotechnological production of alcohols and derivatives thereof

ABSTRACT

The present invention relates to a microorganism having a reduced fatty acid degradation capacity and expressing a recombinant alkane oxidase, a method for oxidizing an alkyl, comprising a contacting the alkyl with an aqueous solution comprising the inventive cell.

The present invention relates to a microorganism having a reduced fatty acid degradation capacity and expressing a recombinant alkane oxidase, a method for oxidizing an alkyl, comprising a contacting the alkyl with an aqueous solution comprising the inventive cell.

Industrial production of bulk and fine chemicals based on renewably resources such as biomass has a range of shortcomings. One of them is the need to extract from a large volume of aqueous medium the product of interest, a procedure necessary not only to concentrate the product for further downstream processing but also to limit its concentration in an aqueous medium typically comprising live cells. Such cells are extremely potent but nonetheless delicate catalysts which cannot be subjected to harsh conditions, for example high temperatures, extreme values of pH, the presence of hazardous solvents, products and the like, or else there is the chance that they might lyse to the effect that their catalytic activity is lost and cell debris, metabolites and macromolecules are released that may contaminate or even degrade the sought-after product.

In order to extract the product of interest, the aqueous medium is typically contacted with a comparably small volume of a water-immiscible organic solvent. As a result, a product having sufficiently high a degree of hydrophobicity is released from the aqueous culture medium and accumulates in the water-immiscible solvent. The product may subsequently be subjected to further synthetic steps compatible with the water-immiscible solvent or may be purified, for example by way of distillation or crystallization.

Whether or not a compound may be extracted from an aqueous phase using a water-immiscible organic solvent depends on its physicochemical properties. While compounds rich in or entirely consisting of unsubstituted carbon chains are likely to enter the solvent, compounds having functional groups comprising heteroatoms or even charges should be expected to prevail in the aqueous phase.

A compound's relative distribution in a liquid biphasic system which has reached the equilibrium state may be described using the Nernst distribution law:

α=c _(Phase 1) /c _(Phase 2),

wherein c₁ and c₂ are the molar equilibrium concentrations of the compound in the first and second phase, respectively, and the constant k is a temperature-dependent distribution coefficient. The distribution properties of a compound may also described using the K_(ow) or P value, in case the biphasic system comprises an aqueous phase and a phase comprising octanol:

K _(ow) =P=c _(Octanol) /c _(Water).

These equations describe the distribution of a compound in a given liquid biphasic system, however, they apply only when the distribution equilibrium has been reached. If pure water and a pure water-immiscible organic solvent, for example water and hexane, are mixed, two distinct phases will emerge almost instantly. However, the situation is very different if an aqueous culture medium comprising a live cell and a water-immiscible solvent are contacted. Owing to the numerous possible molecular interactions, separation of the water-immiscible solvent may take several hours if not days; meanwhile the cells are subjected to contact with the potentially toxic solvent. Hence, the time taken for the biphasic state to be reached is a parameter that should be optimised if an efficient process for the biotechnological production of chemicals is to be devised.

Substituted alkanes, for example alcohols, aldehydes, ketones, carboxylic acids and amines, represent of a class of industrially sought-after compounds traditionally prepared by conversion of compounds made from fossil carbon sources. In an era of increasingly limiting supplies of non-renewable fossil fuels, there is considerable interest in biotechnological processes for producing alkanes and derivates thereof starting with renewable resources, i.e. materials that are easily and, in terms of geological time scales, rapidly replenishable.

Numerous methods for converting an alkane into a corresponding substituted alkane, in particular an oxidised alkanes, have been reported in the prior art. Methane monooxygenases catalyse the NADH-dependent insertion of one atom of oxygen into the C—H bond of methane to form methanol, the first step in the degradation of methane by methanotrophs such as Methylosinus trichosporium and Methylococcus capsulatus. The soluble methane monooxygenases typically have a broad substrate spectrum including saturated and unsaturated, linear, branched, and cyclic hydrocarbons up to about C8, as well as aromatic, heterocyclic, and chlorinated compounds (Merkx M, Kopp D A, Sazinsky M H, Blazyk J L, Müller J, Lippard S J (2001), Angew Chem Int Ed Engl 40:2782-2807; Higgins I J, Best D J, Hammond R C. 1980. New findings in methane-utilizing bacteria highlight their importance in the biosphere and their commercial potential. Nature 286:561-564.). Heme-containing oxygenases, most notably those from the class of cytochrome P450 systems including cytochrome P450 BM-3 from Bacillus megaterium, also use molecular oxygen in order to hydroxylate alkanes of various carbon chain lengths and have been subjected to protein evolution approaches (Koch D J, Chen, M M, van Beilen, J. B. and Arnold F. H. (2009, Appl. And Environm. Microbiol. 75(2), 337-344). Rubredoxin-dependent alkane monoxygenases such as the alkane monooxygenase from Pseudomonas putida GPo1 catalyse the oxidation of alkanes of medium chain lengths, yielding a mixture of alcohols and carboxylic acids (Grant C., Woodley, J. M, and Baganz, F (2011) Enzyme and Microbial Technology 48, 480-486). Xylene monoxygenases hydroxylate alkanes which may subsequently be converted to various other substituted alkanes including amines, carboxylic acids, amides, alkyl halides, esters, alkenes, using biotechnological or synthetic approaches (Bruce, P. Y. (1998), Organic Chemistry, Sec. Ed., Prentic Hall Inc.).

Alkanes consist only of hydrogen and carbon atomes bonded by single bonds and as such lack functional groups comprising heteroatoms. Consequently, even short alkanes substituted with a polar functional group, let alone those comprising long unsubstituted carbon chains, are soluble in water-immiscible organic solvents. Last but not least, many of them, for example methanol and ethanol, are reactive and are known to have adverse effects on the growth, viability and metabolism of biotechnologically relevant microorganisms. Therefore, many biotechnological processes for the production of such compounds comprise an extraction step using water-immiscible solvents.

Therefore, the problem underlying the present invention is to provide a biotechnological process for converting an alkane to a substituted alkane in a biphasic system comprising an aqueous medium and a water-immiscible solvent, wherein the separation of the water-immiscible solvent from the aqueous medium is rapid. More specifically, the problem underlying the present invention is to improve the separation of a water-immiscible solvent and a substituted alkane solved therein in terms of the time taken to separate the two phases, the degree of separation of the solvent in a given time, yield and purity of the product solved therein or obtained following further processing as well as viability and/or recyclability of any cells contacted with the water-immiscible solvent.

Another problem underlying the present invention is to provide a cell that may be used for such a process, preferably one that is more resistant towards stress caused by the presence of water-immiscible organic solvents, for example in terms of oxygen consumption, oxygen consumption per amount of product, growth rates, metabolic activity and viability in general.

Another problem underlying the present invention is to set up a process for producing oxidation products of alkanes and derivatives thereof based and sustainable resources.

Another problem underlying the present invention is to provide a biotechnological process for oxidizing alkanes, wherein the consumption of oxygen is reduced.

The problem underlying the present invention is solved by the subject matter of the attached claims.

The problem underlying the present invention is solved, in a first aspect, by a microorganism having a reduced fatty acid degradation capacity and expressing a recombinant alkane oxidase.

In a first embodiment of the first aspect, the alkane oxidase is selected from the group comprising rubredoxin-dependent alkane oxidases, cytochrome P450 alkane oxidases, xylene monooxygenases, methane monoxygenases and variants thereof.

In a second embodiment, which is also an embodiment of the first embodiment of the first aspect of the present invention, the fatty acid degradation capacity is reduced by deletion of a gene encoding an enzyme from the group comprising fatty acid importer, fatty acid-CoA ligase, acyl-CoA dehydrogenase, 2,4-dienoyl-CoA reductase, enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase.

In a third embodiment, which is also an embodiment of the first and the second embodiment of the first aspect of the present invention, the microorganism is a prokaryotic or a lower eukaryotic cell, preferably a bacterial cell, most preferably E. coli.

In a fourth embodiment, which is also an embodiment of the first to third embodiments of the first aspect of the present invention, the microorganism further expresses a recombinant alcohol dehydrogenase.

In a fifth embodiment, which is also an embodiment of the first to fourth embodiments of the first aspect of the present invention, the microorganism further expresses a recombinant transaminase.

In a sixth embodiment, which is also an embodiment of the first to fifth embodiments of the first aspect of the present invention, the microorganism expresses a recombinant amino acid dehydrogenase, preferably an alanine dehydrogenase.

The problem underlying the present invention is solved, in a second aspect, by a method for oxidising an alkyl, comprising a) contacting the alkyl with an aqueous solution comprising the cell according to the first aspect of the present invention and any of its embodiments.

In a first embodiment of the second aspect of the present invention, the problem is solved by a method further comprising b) contacting the aqueous solution from step a) with a water-immiscible organic solvent.

In a second embodiment of the second aspect of the present invention which is also an embodiment of the first embodiment, the extraction is carried out following completion of the alkyl oxidation and preferably removal of the inventive cell from the aqueous solution.

The problem underlying the present invention is solved, in a third aspect, by a use of the microorganism according to the first aspect and any of its embodiments for oxidising an alkyl.

In a further embodiment of the second or third aspect or any of its embodiments, the alkyl is a compound represented by the formula H—(CH₂)_(x)—R, wherein x is 1 to 30, and R is any chemical group, preferably one selected from the group comprising —OH, —COH, —COOH, —COOR¹, —NH₂, —NO₂, —CN, —OPO₃H, —SO₃H and —H, and is preferably H, and wherein R¹ is an unsubstituted linear alkyl, preferably methyl and ethyl.

In a further embodiment of the second or third aspects or any of their embodiments, the alkyl is a linear alkane, preferably one that is gaseous at room temperature.

In a further embodiment of the second or third aspects of the invention and any of their embodiments, the water-immiscible organic solvent is a water-immiscible fatty acid or fatty acid ester.

The inventors of the present invention have surprisingly found that the separation of a water-immiscible organic solvent from an aqueous culture medium comprising a microorganism capable of catalyzing alkane or alkyl oxidation is more rapid and complete if a microorganism having a reduced fatty acid degradation capacity rather than a corresponding wild type microorganism is used.

Moreover, the present inventors have found that microorganisms having a reduced fatty acid degradation capacity consume less oxygen relative to the respective wild type microorganisms while the yield of the product is equal or even improved.

Without wishing to be bound by any theory, the inventors of the present invention theorise that reducing the fatty acid degradation capacity of a cell leads to lowered levels of at least one as yet unidentified metabolite acting as a detergent and counteracting separation of a water-immiscible organic solvent, located either intracellularly or at the surface of the cell of interest.

The present invention contemplates a method for oxidising an alkyl. The alkyl may be oxidised to one or more compounds selected from the group comprising an alcohol, an aldehyde, a ketone, and a carboxylic acid. Such a compound may be the main product of an inventive process, but it may also be further processed. For example, the method may not only comprise the oxidation of an alkyl to a carboxylic acid, but also the conversion of such a carboxylic acid to an amide.

The present invention centers around a microorganism having a reduced fatty acid degradation capacity and expressing a recombinant alkane oxidase, preferably for the conversion of alkyls to oxidized alkyls such as alcohols. In a preferred embodiment, the term “alkane oxidase”, as used herein, refers to any enzyme capable of oxidizing an alkane and/or alkyls. Various alkane oxidases have been described in the literature, for example basidiomycete heme-thiolate peroxidases (Gutierrez, A., Babot, E. D., Ullrich, R., Hofrichter, M., Martinez, A. T., del Rio, J. C. (2011), Arch. Biochem. Biophys. 514 (1-2), 33-43), the alkane hydroxylase system of Gardonia sp. strain SoCg (Lo Piccolo, L., De Pasquale, C., Fodale, R., Puglia, A. M., Quatrini, P. (2011), Appl. Environm. Microbiol. 77 (4), 1204-12013; Alkane oxidases from Alcanivorax (Grant, C., Woodley, J. M., Baganz, F. (2011) Enzyme and Microbial Technology, 480-486) and Cytochrom P450 systems (Koch, D. J., Chen, M. M., van Beilen, J. B., and Arnold F. H. (2009) Appl. and Env. Microbiology, 337-344). In a particularly preferred embodiment, the alkane oxidase is an alkB type alkane oxidase. AlkB is an oxidoreductase from the Pseudomonas putida AlkBGT system, dependent on two auxiliary polypeptides, AlkG and AIkT. AIkT is a FAD-dependent rubredoxin reductase transferring electrons from NADH to AlkG. AlkG is a rubredoxin, an iron-containing redox protein functioning as a direct electron donor to AlkB. In a preferred embodiment, the term “alkB type alkane oxidase”, as used herein, refers to AlkB from Pseudomonas putida Gpo1 (Access code: CAB54050.1, any access code used in the application refers to the respective sequence from the Genbank database run by the NCBI, wherein the release referred to is the one online on the 15 Dec. 2011) or a variant thereof. In a preferred embodiment, the term “alkyl” refers to a substituent represented by the formula H—(CH₂)_(x)—R, wherein R is any chemical group and x is 1, 2, 3, . . . , preferably 8 or more, more preferably 11 or more, or to a compound comprising such a substituent.

In a preferred embodiment, the term “rubredoxin-dependent alkane oxidase”, as used herein refers to an oxidoreductase that recognises as its substrate an alkane receiving electrons via a rubredoxin, the latter being, in a more preferred embodiment, an iron-sulphur protein having an α+β class fold with 2 α helices and 2 to 3 β-strands transferring electrons to the alkane oxidase and is, in a most preferred embodiment, AlkG from Pseudomonas putida or a variant thereof. Examples include AlkG from Pseudomonas putida.

In a preferred embodiment, the term “cytochrome P450 enzyme”, as used herein, refers to an oxidoreductase having a P450-type cytochrome having, in its CO-bound form an absorption band at 450 nm and preferably capable of oxidizing an alkane. Examples include cytochrome P450 BM-3 from Bacillus megaterium (Koch, D. J., Chen, M. M., van Beilen, J. B., and Arnold, F. H. (2009) Appl. And Environm. Microbiol. 75(2), 337-344).

In a preferred embodiment, the term “xylene monooxygenase”, as used herein, refers to a membrane-spanning, non-heme diiron enzyme oxidoreductase with a histidine-rich active site and preferably capable of oxidizing an alkane. Examples include XylM from Pseudomonas putida (Austin, R. N., Buzzi, K., Kim, E., Zylstra, G. J., and Groves, J. T. (2003) J. Biol. Inorg. Chem. 8, 733-740.

In a preferred embodiment, the term “methane monooxygenase”, as used herein, refers to an oxidoreductase that is either a soluble methane monooxygenase comprising di-iron center bridged by an oxygen atom (Fe—O—Fe) and comprising three protein components, a hydroxylase, a β unit, and a reductase, from a methanotropic bacterium or is a particulate methane monooxygenase, a membrane-protein in a methanotrophic bacterium, comprising a copper-containing active site. Examples of soluble and particulate methane monooxygenases comprises the soluble methane monooxygenase from Methylosinus trichosporium OB3b (A C Rosenzweig, Frederick, C. A., Lippard, S. J., Nordlung, P. (1993) Nature, 366, 537-543) and the particulate methane monooxygenase from Methylococcus capsulatus (Bath) (Nguyen, H. H. T., Elliot, S. J., Yip, J. H. K, and Chan, S. I. (1998), J. Biol. Chem. 273, 7957-7966), respectively.

The teachings of the present invention may not only carried out using biological macromolecules having the exact amino acid or nucleic acid sequences referred to in this application explicitly, for example by name or accession number, or implicitly, but also using variants of such sequences. In a preferred embodiment, the term “variant”, as used herein, comprises amino acid or nucleic acid sequences, respectively, that are 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to the reference amino acid or nucleic acid sequence, wherein preferably amino acids other than those essential for the function, for example the catalytic activity of a protein, or the fold or structure of a molecule are deleted, substituted or replaced by insertions or essential amino acids are replaced in a conservative manner. The state of the art comprises algorithms that may be used to align two given nucleic acid or amino acid sequences and to calculate the degree of identity, see Arthur Lesk (2008), Introduction to bioinformatics, 3^(rd) edition, Thompson et al., Nucleic Acids Research 22, 4637-4680, 1994, and Katoh et al., Genome Information, 16(1), 22-33, 2005. The term “variant” is used synonymously and interchangeably with the term “homologue”. Such variants may be prepared by introducing deletions, insertions or substitutions in amino acid or nucleic acid sequences as well as fusions comprising such macromolecules or variants thereof. In a preferred embodiment, the term “variant”, with regard to amino acid sequence, comprises, preferably in addition to the above sequence identity, amino acid sequences that comprise one or more conservative amino acid changes with respect to the respective reference or wild type sequence or comprises nucleic acid sequences encoding amino acid sequences that comprise one or more conservative amino acid changes. In a preferred embodiment, the term “variant” of an amino acid sequence or nucleic acid sequence comprises, preferably in addition to the above degree of sequence identity, any active portion and/or fragment of the amino acid sequence or nucleic acid sequence, respectively, or any nucleic acid sequence encoding an active portion and/or fragment of an amino acid sequence. In a preferred embodiment, the term “active portion”, as used herein, refers to an amino acid sequence or a nucleic acid sequence, which is less than the full length amino acid sequence or codes for less than the full length amino acid sequence, respectively, wherein the amino acid sequence or the amino acid sequence encoded, respectively retains at least some of its essential biological activity. For example an active portion and/or fragment of a protease is capable of hydrolysing peptide bonds in polypeptides. In a preferred embodiment, the term “retains at least some of its essential biological activity”, as used herein, means that the amino acid sequence in question has a biological activity exceeding and distinct from the background activity and the kinetic parameters characterising said activity, more specifically k_(cat) and K_(M), are preferably within 3, more preferably 2, most preferably one order of magnitude of the values displayed by the reference molecule with respect to a specific substrate. In a preferred embodiment, the term “variant” of a nucleic acid comprises nucleic acids the complementary strand of which hybridises, preferably under stringent conditions, to the reference or wild type nucleic acid. Stringency of hybridisation reactions is readily determinable by one of ordinary skilled in the art, and in generally is an empirical calculation dependent on probe length, washing temperature and salt concentration. In general longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridisation generally depends on the ability of denatured DNA to reanneal to complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridisable sequence, the higher the relative temperature which may be used. As a result it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperature less so. For additional details and explanation of stringency of hybridisation reactions, see F. M. Ausubel (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Moreover, the person skilled take in the art may follow the instructions given in the manual “The DIG System Users Guide for Filter Hybridization”, Boehringer Mannheim GmbH, Mannheim, Germany, 1993 and in Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991) on how to identify DNA sequences by means of hybridisation. In a preferred embodiment, stringent conditions are applied for any hybridisation, i.e. hybridisation occurs only if the probe is 70% or more identical to the target sequence. Probes having a lower degree of identity with respect to the target sequence may hybridise, but such hybrids are unstable and will be removed in a washing step under stringent conditions, for example lowering the concentration of salt to 2×SSC or, optionally and subsequently, to 0.5×SSC, while the temperature is, in order of increasing preference, approximately 50° C.-68° C., approximately 52° C.-68° C., approximately 54° C.-68° C., approximately 56° C.-68° C., approximately 58° C.-68° C., approximately 60° C.-68° C., approximately 62° C.-68° C., approximately 64° C.-68° C., approximately 66° C.-68° C. In a particularly preferred embodiment, the temperature is approximately 64° C.-68° C. or approximately 66° C.-68° C. It is possible to adjust the concentration of salt to 0.2×SSC or even 0.1×SSC. Polynucleotide fragments having a degree of identity with respect to the reference or wild type sequence of at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% may be isolated. In a preferred embodiment, the term “homologue” of a nucleic acid sequence, as used herein, refers to any nucleic acid sequence that encodes the same amino acid sequence as the reference nucleic acid sequence, in line with the degeneracy of the genetic code.

The inventive teachings may be carried out using a wide range of microorganisms. In a preferred embodiment, the term “microorganism”, as used herein, refers to any permanently unicellular microorganism comprising bacteria archaea, fungi, algae and the like. In a preferred embodiment, the microorganism is a bacterial microorganism, more preferably one from the group comprising Pseudomonas, Corynebacterium and Escherichia, most preferably Escherichia coli. In another preferred embodiment, the microorganism is a lower eukaryote, more preferably a fungi from the group comprising Saccharomyces, Candida, Picchia, Schizosaccharomyces and Yarrowia, and is most preferably Saccharomyces cerivisiae. Throughout this application, the term “microorganism” is used synonymously and interchangeably with the term “cell”. The microorganism may be an isolated microorganism, in other words a pure culture of a single strain of microorganism, or may comprise a mixture of at least two strains. Biotechnologically relevant microorganisms are commercially available, for example from the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSMZ). Particles for keeping and modifying microorganisms are available from the prior art, for example Sambroke/Fridge/Maniadis (1989): Molecular cloning—A Laboratory Manual, Cold Spring Harbour Press, 2^(nd) edition, Fuchs/Schlegel (2007), Allgemeine Mikrobiologie, 2008, Georg Thieme Verlag.

The inventive microorganism has a reduced fatty acid degradation capacity. In a preferred embodiment, the term “having a reduced fatty acid degradation capacity”, as used in herein, means that the respective microorganism degrades fatty acids, preferably those taken up from the environment, at a lower rate than a comparable microorganism having normal fatty acid degradation capacity would. In a preferred embodiment, the fatty acid degradation of such a microorganism is lower on account of deletion, inhibition or inactivation of at least one gene encoding an enzyme involved in the β-oxidation pathway. In a preferred embodiment of the present invention, at least one enzyme involved in the β-oxidation pathway has lost, in order of increasing preference, 5, 10, 20, 40, 50, 75, 90 or 99% activity relative to the activity of the same enzyme under comparable conditions in the respective wild type microorganism. The person skilled in the art is familiar with various techniques that may be used to delete a gene encoding an enzyme or reduce the activity of such an enzyme in a microorganism, for example by exposition of cells to radioactivity followed by accumulation or screening of the resulting mutants, site-directed introduction of point mutations or knock out of a chromosomally integrated gene encoding for an active enzyme, as described in Sambrook/Fritsch/Maniatis (1989). In addition, the transcriptional repressor FadR may be over expressed to the effect that expression of enzymes involved in the β-oxidation pathway is repressed (Y Fujita, H Matsuoka, and K Hirooka (2007) Mol. Microbiology 66(4), 829-839). In a preferred embodiment, the term “deletion of a gene”, as used herein, means that the nucleic acid sequence encoding said gene is modified such that the expression of active polypeptide encoded by said gene is reduced. For example, the gene may be deleted by removing in-frame a part of the sequence comprising the sequence encoding for the catalytic active centre of the polypeptide. Alternatively, the ribosome binding site may be altered such that the ribosomes no longer translate the corresponding RNA. Moreover, the person skilled in the art is able to routinely measure the activity of enzymes expressed by living cells using standard essays as described in enzymology text books, for example A Cornish-Bowden (1995), Fundamentals of Enzym Kinetics, Portland Press Limited, 1995. The state of the art discloses various tests designed specifically for determining the activity of enzymes involved in the β-oxidation pathway, for example K Kameda & W D Nunn (1981) J. Biol. Chem. 256, 5702-5707, H Marrakchi, W E DeVVolf, C Quinn, J West, B J Polizzi, C Y So et al. (2003) Biochem. J. 370, 1055-1062, S Lobo, G Florova, and K A Reynolds (2001) Biochemistry 40 (39), 11955-64, X Yu, T Liu, F Zhu, and C Khosla (2011) PNAS, published online).

Degradation of fatty acids in microorganisms is accomplished by a sequence of enzymatically catalysed reactions. First of all, fatty acids are taken up and translocated across the cell membrane via a transport/acyl-activation mechanism involving at least one outer membrane protein and one inner membrane-associated protein which has fatty acid-CoA ligase activity, referred to in the case of E. coli as FadL and FadD, respectively. Inside the cell, the fatty acid to be degraded is subjected to enzymes catalyzing other reactions of the β-oxidation pathway. The first intracellular step involves the conversion of acyl-CoA to enoyl-CoA through acyl-CoA dehydrogenase, the latter referred to as FadE in the case of E. coli. The resulting enoyl-CoA is converted to 3-ketoacyl-CoA via 3-hydroxylacyl-CoA through hydration and oxidation, catalysed be enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, referred to as FadB in E. coli. Finally, 3-ketoacyl-CoA thiolase, FadA in E. coli, catalyses the cleaveage of 3-ketoacyl-CoA, to give acetyl-CoA and the input acyl-CoA shortened by two carbon atoms. In a preferred embodiment, the term “a microorganism having a reduced fatty acid degradation capacity”, as used herein, refers to a microorganism having a reduced capability of taking up and/or degrading fatty acids, preferably those having at least eight carbon chains. The fatty acid degradation capacity of a microorganism may be reduced in various ways. In a preferred embodiment, the microorganism has, compared to its wild type, a reduced activity of an enzyme involved in the β-oxidation pathway. In a preferred embodiment, the term “enzyme involved in the β-oxidation pathway”, as used herein, refers to an enzyme that interacts directly with a fatty acid or a derivative thereof formed as part of the degradation said fatty acid via the β-oxidation pathway the sequence of reactions effecting the conversion of a fatty acid to acetyl-CoA and the CoA ester of the shortened fatty acid, preferably by recognizing the fatty acid or derivative thereof as a substrate, and converts it to a metabolic formed as a part of the β-oxidation pathway. In a particularly preferred embodiment, the term “enzyme involved in the β-oxidation pathway” includes a fatty acid importer, more specifically any component of the fatty acid import machinery such as FadL or variants thereof and a membrane-bound fatty acid CoA-ligase. For example, the acyl-CoA dehydrogenase is an enzyme involved in the β-oxidation pathway as it interacts with fatty acid-CoA and converts fatty acid-CoA ester to enoyl-CoA, which is a metabolite formed as part of the β-oxidation. In a preferred embodiment, the term “enzyme involved in the fatty inter oxidation pathway”, as used herein, comprises any polypeptide from the group comprising fatty acid importer and components thereof, fatty acid CoA-ligase, acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacly-CoA dehydrogenase and 3-keto-acyl-CoA thiolase. In a preferred embodiment, the term “fatty acid transporter”, as used herein, refers to a polypeptide capable of translocating a fatty acid from the outer side of the membrane or outer, i.e. medium-exposed membrane, membrane of a microorganism into the inside of the cell, possibly as part of a machinery comprising several active polypeptides. For example, the polypeptide FadL (access code: BAA16205.1) in E. coli is a fatty acid transporter. In a preferred embodiment, the term “fatty acid-CoA ligase”, as used herein, refers to a polypeptide capable of catalysing the conversion a fatty acid to the CoA ester of a fatty acid, i.e. a molecule, wherein the functional group —OH of the carboxy group is replaced with —S-CoA, preferably for introducing said fatty acid into the β-oxidation pathway. For example, the polypeptide FadD in E. coli (access code: BAA15609.1) is an acyl-CoA dehydrogenase. In a preferred embodiment, the term “acyl-CoA dehydrogenase”, as used herein, is a polypeptide capable of catalysing the conversion of an acyl-CoA to enoyl-CoA, preferably as part of the β-oxidation pathway. For example, the polypeptide FadE in E. coli (access code: BAA77891.2) is an acyl-CoA dehydrogenase. In a preferred embodiment, the term “2,4-dienoyl-CoA reductase”, as used herein, is a polypeptide capable of catalysing the conversion of the 2,4-dienoyl CoA from an unsaturated fatty acid into enoyl-CoA, preferably as part of the β-oxidation pathway. For example, the polypeptide FadH in E. coli is a 2,4-dienoyl-CoA reductase. In a preferred embodiment, the term “enoyl-CoA hydratase”, as used herein, also referred to as 3-hydroxyacyl-CoA dehydrogenase, refers to a polypeptide capable of catalysing the conversion of enoyl-CoA to 3-ketoacyl-CoA through hydration and oxidation, preferably as part of the β-oxidation pathway. For example, the polypeptide FadB in E. coli (access code: BAE77457.1) is an enoyl-CoA hydratase. In a preferred embodiment, the term “ketoacyl-CoA thiolase”, as used herein, refers to a polypeptide capable of catalysing the conversion of cleaving 3-ketoacyl-CoA, resulting in an acyl-CoA shortened by two carbon atoms and acetyl-CoA, preferably as the final step of the b-oxidation pathway. For example, the polypeptide FadA in E. coli (access code: AP009048.1) is a ketoacyl-CoA thiolase.

A multitude of compounds may be converted to an alcohol using the inventive microorganism, as long as they comprise an alkyl substituent, including, but not limited to alkanes, alkenes, alkynes, aryls, heteroaryls, alcohols, amines, alkanoic acids, alkenoic acids, lipids, amino acids, saturated or unsaturated and/or linear or branched fatty acids. In a preferred embodiment, the term “alkyl”, as used herein, is a compound represented by the formula H—(CH₂)_(x)—R, wherein x is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28 or, in order of increasing preference, at least 6, 8, 10 or 12, and R is any chemical group, preferably one selected from the group comprising —OH, —COH, —COOH, —COOR¹, wherein R¹ is an unsubstituted linear alkyl, preferably Methyl and Ethyl, —NH₂, —NO₂, —CN, —OPO₃H, —SO₃H and —H, and is preferably H. In a preferred embodiment, the alkyl is a fatty acid or an ester thereof. In an other preferred embodiment, the alkyl is an alkane represented by the formula C_(n)H_(2n+2) including branched alkanes, cycloalkanes and cycloalkanes having one or more linear alkyl substituents, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and preferably 1 to 12, more preferably 1 to 4. In another preferred embodiment, the alkyl is an alkyl gaseous at 25° C. and under atmospheric pressure, including branched alkyls. In a preferred embodiment the alkane is isobutane.

It may be advantageous to use a microorganism that has, in addition to the recombinant alkane oxidase, further enzymes, preferably recombinant enzymes. In a preferred embodiment, the microorganism has, in addition to the alkane oxidase, a wild type or, preferably recombinant, alcohol dehydrogenase. In a preferred embodiment, the term “alcohol dehydrogenase”, as used herein, refers to an enzyme capable of catalyzing the conversion of an alcohol to the corresponding aldehyde or ketone. Examples include, but are not limited to the alcohol dehydrogenases from Bacillus stearothermophilus (access code P42328), Rhodococcus ruber (access code AJ491307.1), Ralstonia eutropha (access code ACB78191.1), Lactobacillus brevis (access code YP_(—)795183.1), Lactobacillus kefiri (access code ACF95832.1), Paracoccus pantotrophus (access code ACB78182.1) and Sphingobium yanoikuyae (access code EU427523.1) as well as variants thereof.

The inventive microorganism may have, in addition to an alkane oxidase and an alcohol dehydrogenase, a transaminase, preferably a recombinant transaminase, which is advantageous if the aim is to convert an alkyl into an amine. In a preferred embodiment, the term “transaminase”, as used herein, refers to an enzyme capable of transferring α-amino groups from a donor, preferably an amino acid, to an acceptor molecule, preferably an α-keto acid, to yield another amino acid. In a particularly preferred embodiment, the transaminase is a ω-transaminase. Examples of transaminases include, but are not limited to the transaminase from Chromobacterium violaceum ATCC 12472 (access code NP_(—)901695).

The inventive microorganism may have, in addition to an alkane oxidase, an alcohol dehydrogenase and a transaminase, an amino acid dehydrogenase preferably a recombinant amino acid dehydrogenase. In a preferred embodiment, the term “alanine dehydrogenase”, as used herein, refers to an enzyme capable of catalysing the conversion of an amino acid, water and NAD to a keto acid, ammonia and NADH. The amino acid dehydrogenase may be an alanine dehydrogenase, i.e. an enzyme capable of catalysing the conversion of L-alanine, water and NAD to pyruvate, ammonia and NADH. Examples of suitable amino acid dehydrogenases comprise alanine dehydrogenases from Bacillus subtilis (access code L20916), Rhizobium leguminosarum (access code: CP001622), Vibrio proteolytikus (access code: AF070716), Mycobacterium tuberculosis (access code: X63069) and Enterobacter aerogenes (access code AB013821).

The inventive method comprises contacting the alkyl with an aqueous solution comprising the inventive cell. This step may not only comprise temporarily contacting the alkyl with the solution, but in fact incubating the alkyl in the presence of the inventive cell sufficiently long to allow for an oxidation reaction and possible further downstream reactions to occur, for example for at least 1, 2, 4, 5, 10 or 20 hours. The temperature chosen must be such that the inventive cells remains catalytically competent and/or metabolically active, for example 10 to 42° C., preferably 30 to 40° C., most preferably 32 to 38° C. in case the inventive cell is an E. coli cell.

In an embodiment, the inventive method contemplates contacting the product from step a) using a “water-immiscible organic solvent” following step a) or simultaneous with step a). The person skilled in the art knows numerous water-immiscible organic solvents that may be used according to the present invention. In a preferred embodiment, the term “water-immiscible organic solvent”, as used herein, refers to a compound comprising at least two carbon atoms and having the tendency to form, in the presence of an aqueous liquid phase, preferably at 25° C., another liquid phase, which is clearly separate from the aqueous phase. The separate phase may be a continuous liquid phase or an emulsion. In another preferred embodiment, the term “water-immiscible”, as used herein, refers to the tendency of a liquid compound not to be soluble in the water. Finally in another preferred embodiment, the term “water-immiscible”, as used herein, means that a compound designated as such has a pH-value (J Sangster, Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry, Vol. 2 of Wiley Series in Solution Chemistry, John Wiley & Sons, Chichester, 1997) the decadic logarithm of which exceeds 0, preferably 0.5, more preferably 1 and most preferably 2. Preferred water-immiscible organic solvents comprise, but are not limited to water-immiscible solvents from the group comprising substituted and linear alkanes liquid at room temperature, cycloalkanes, cycloalkenes, aryls, fatty acids, fatty acid esters, alcohols, heterocycloalkanes, heterocycloalkenes and heteroaryls. The water-immiscible organic solvents may comprise more than one organic solvent. In a preferred embodiment, the term “extracting” a product using a “water-immiscible organic solvent”, as used herein, means that the aqueous solution comprising the inventive cell is contacted with the water-immiscible organic solvent sufficiently long as to allow the product to enter the phase comprising the water-immiscible solvent. Subsequently, the phase comprising the water-immiscible organic solvent may be separated from the aqueous solution, for example by distillation or by decantation. Whether a compound is liquid or gaseous or neither, is preferably determined at 25° C. under atmospheric pressure.

In a preferred embodiment, the water-immiscible organic solvent is a fatty acid or an ester thereof, in a more preferred embodiment a fatty acid represented by the formula CH₃—(CH₂)_(x)—COOR^(S), wherein x is 8, 9, 10, . . . , 28 and is more preferably 12 or more than 12, and wherein R^(S) is H, or alkyl, the latter preferably methyl or ethyl. In another preferred embodiment, the water-immiscible organic solvent is an unsaturated fatty acid, preferably one having a carbon-carbon double bond at position 9 of the carbon chain, more preferably one having 12 carbon atoms or more. In a most preferred embodiment, the water-immiscible organic solvent is oleic acid. In another preferred embodiment, the water-immiscible solvent is hexanoic acid. In a preferred embodiment, the water-immiscible organic solvent is lauric acid methyl ester. The volume of the water-immiscible organic solvent is such that it is straightforward to separate it from the aqueous solution. In a preferred embodiment, the volume of the water-immiscible organic solvent is 2 to 98, more preferably 5 to 95, more preferably 10 to 40, most preferably 20 to 30 percent of the total combined volumes of aqueous solution and water-immiscible organic solvent.

In a preferred embodiment, the term “contacting”, as used herein, means bringing about direct contact between alkyl and inventive cell such that the cell is able to take up and metabolise the alkyl. For example, the cell and the alkyl may not be in different compartments separated by a membrane such as an inorganic membrane. If the alkyl is solid or soluble, it may simply be added to the inventive cell in an aqueous solution. If the alkyl is gaseous, the aqueous solution comprising the cell may be sparged with a gas comprising said gaseous alkyl.

The term “an aqueous solution” comprises any solution that may be used to keep the inventive cell, at least temporarily, in a metabolically active and/or viable state and comprises, if such is necessary, any additional substrates. The person skilled in the art is familiar with numerous aqueous solution, usually referred to as media, that may be used to keep inventive cells, for example LB medium in the case of E. coli. In a preferred embodiment the aqueous solution is kept under aerobic conditions. It is advantageous to use as an aqueous solution a minimal medium, i.e. a medium of reasonable simple composition that comprises only the minimal set of salts and nutrients indispensible for keeping the cell in a metabolically active and/or viable state, by contrast to complex mediums. For example, M9 medium may be used as a minimal medium. If the alkyl to be oxidised has limited solubility in water, a detergent such as Tween or Triton may be added to the aqueous solution or a hydrophobic solvent may be used to solubilise the alkyl to be oxidised. The person skilled in the art is familiar with the preparation of various aqueous and organic solutions.

In a preferred embodiment, step b) is carried out following completion of the alkyl oxidation and preferably removal of the inventive cell from the aqueous solution. In a preferred embodiment, the alkyl oxidation, i.e. the oxidation of the alkyl catalysed by the inventive cell, may be regarded as complete if at least one of the following requirements is met: a) the inventive cell ceases to be metabolically active, b) there is no detectable substrate turnover c) there is no more substrate present in the aqueous solution d) the net amount of product does no longer increase significantly, for example if a concentration plateau has been reached or the slope of the graph indicating product formation is, over any period of two hours, zero or less. Cells may be removed from the aqueous solution in numerous ways known to the person skilled in the art, for example by centrifugation, filtration or decantation.

The invention is further illustrated by the following figures and non-limiting examples from which further features, embodiments, aspects and advantages of the present invention may be taken.

FIG. 1 shows different phase separation behaviour if ΔFadE mutant W3110 ΔFadE [alkB-alaD-TA] (left), also referred to as “ΔFadE”, and strain W3110 [alkB-alaD-TA] (right), also referred to as wild type (VVT), the latter identical to the former strain except for the fact that is FadE is not deleted, are used to produce ALSME. The arrow points the interphase between organic and aqueous phase visible after ten minutes in case the mutant is used. No such interphase is detectable after ten minutes in case the wild type strain is used.

FIG. 2 shows the results of the same experiment as described with respect to FIG. 1, except for the fact that the medium was transferred to Falcon tubes after completion of the fermentation.

FIG. 3 shows the oxygen transfer rate and the carbon dioxide transfer rate of both strains used for the experiment described with respect to FIG. 1.

FIG. 4 shows the concentrations of ALSME over time in the same experiment described with respect to FIG. 1.

EXAMPLE 1 Acceleration of Separation of a Hydrophobic Phase from an Aqueous Medium Using a Cell with Reduced Acyl CoA-Dehydrogenase Activity for the Production of ω-Amino Lauric Acid Methyl Ester (ALSME)

The conversion of lauric acid methyl ester to w-amino lauric acid (ALS) methyl ester, via ω-hydroxy lauric acid, was carried out in a parallel fermentation system comprising 8 vessels from DASGIP, using strains W3110 ΔFadE [alkB-alaD-TA] and W3110 [alkB-alaD-TA].

N. B. that these two strains comprise a pBR322-derived plasmid comprising oxidoreductase AlkB, an alcohol dehydrogenase and a transaminase in line with international application WO 2009/077461 and are identical except for the fact that the former has a deletion in the gene encoding FadE, the E. coli acyl-CoA dehydrogenase of the β-oxidation pathway.

1 liter reaction vessels were used for the fermentation. pH electrodes were calibrated by a two-point-calibration using pH 4 and pH 7 standard solutions. Reactors containing 300 mL tap water were autoclaved for 20 minutes at 121° C. Subsequently the pO2-detectors were polarized at the DASGIP system over night (for at least 6 hours). The next morning water was removed under a clean Bench and replaced by 300 mL of high cell density medium complemented with 100 mg/L ampicillin. Subsequently, pO2 detectors were subjected to one-point-calibration (stirrer: 400 rpm, gas flow: 10 sL/h air), and the tubings associated with the feed, correction agent and induction were cleaned by clean in Place using 70% ethanol, followed by 1 M NaOH, followed by rinsing with sterile VE water.

ALS and ALSME producing strains of E. coli were inoculated from the respective cryo cultures in LB medium (25 mL in a 100 mL flask with baffles) complemented with 100 mg/L ampicillin over night at 37° C. and 200 rpm for approximately 18 hours. Subsequently, 2 mL each of the cultures in high cell density medium (glucose 15 g/L (30 mL/L of a separately autoclaved 500 g/L stock solution comprising 1% MgSO₄*7H₂O and 2.2% NH₄Cl), (NH₄)₂SO4 1.76 g/L, K₂HPO₄ 19.08 g/L, KH₂PO₄ 12.5 g/L, yeast extract 6.66 g/L, trisodium dihydrate 2.24 g/L, ammonium ter iron citrate solution: 17 mL/L of a separately autoclaved 1% stock solution, trace element solution: 5 mL/L of a separately autoclaved stock solution (HCl (37%) 36.50 g/L, MnCl₂*4H₂O 1.91 g/L, ZnSO₄*7H₂O 1.87 g/L, ethylenediamintetraacetic acid dihydrate 0.84 g/L, H₃BO₃ 0.30 g/L. Na₂MoO₄*2H₂O 0.25 g/L, CaCl₂*2H₂O 4.70 g/L, FeSO₄*7H₂O 17.80 g/L, CuCl₂*2H₂O 0.15 g/L)) (20 mL per strain in a 100 mL flask with bethels) with 100 mg/L ampicillin were inoculated and incubated at 37° C./200 rpm for another 5.5 hours.

The optical density of a culture at 600 nm was determined in the case of W3110 ΔFadE [alkB-alaD-TA] as 6.9 and 7.4 in the case of W3110 [alkB-alaD-TA]. In order to inoculate the reaction vessels to a final optical density of 0.1, 4.0 mL or 4.4 mL, respectively, were transferred into a 5 mL syringe under sterile conditions and used to inoculate the reaction using a hollow needle and a septum covered by a layer of 70% ethanol. The following standard program was used

DO-controller pH-controller Preset 0% Preset 0 ml/h P 0.1 P 5 Ti 300 s Ti 200 s Min 0% Min 0 mIL/h Max 100%  Max 40 mL/h N XO2 (I gas F (gas (Rotation) from to mixture) from to flow) from to growth and 0% 30% growth and 0% 100% growth 15% 80% biotransformation 400 rpm 1500 rpm biotransformation 21% 21% and 6 sL/h 72 sL/h biotransformation script trigger sharp 31% DO (1/60 h) induction 2 h after feed IPTG start feed trigger 50% DO feed rate 3 [mL/h]

The experiment carried out falls into to phases: the growth phase, wherein the aim is to attain cells at a certain optical density, and the subsequent biotransformation phase, wherein the aim is to convert the substrate lauric acid methyl ester to w-amino lauric acid methyl ester. pH values were maintained at 6.8 using ammonia (12.5%). During culture and biotransformation the dissolved oxygen in the culture was maintained via the stirrer and the gas flow rate at 30%.

The fermentation was carried out as a fed batch, wherein the feed start, 5 g/Lh glucose feed (500 g/L glucose comprising 1% MgSO₄*7H₂O and 2.2% NH₄Cl), was triggered by a DO-Peak. At the time of feed start the temperature was lowered from 37° C. to 30° C. Expression of the transaminase was induced by automatic addition of IPTG (1 mM) 2 h after feed start. alk-genes were induced by manual addition of DCPK (0.025% v/v) 10 h after feed start. The optical density of the culture broth was determined prior to starting the biotransformation.

The biotransformation phase was started 14 h after feed start by adding as a batch a mixture comprising lauric acid methyl ester and oleic acid (technical grade, 90%) to the fermentation broth. In order to provide an amino group donor for the transaminase, half an hour prior to start of the biotransformation 5 mL of a 3M ammonium sulfate solution was added to the fermentation broth. 2 mL fermentation broth samples were removed from the vessel and part of it was diluted 1:20 in a mixture comprising acetone and HCl (c(HCl) 0.1 mol/L) and extracted. Samples were taken 1, 2, 3, 4, 5, 7.5, 10.5, 19.5 and 21 h following start of the biotransformation from all reaction vessels. Oxygen transfer rate (OTR) and carbon transfer rate (CTR) were determined during the fermentation via analysis of exhaust gas from the DASGIP systems. Fermentation was terminated 21 h after start of the biotransformation. The stirrer, the gas flow, the temperature control and pH control were switched of and the vessel was given the opportunity to settle for another 5-10 minutes.

Results:

As the biotransformation progresses, the oxygen and carbon transfer rates increase significantly in the case of W3110 [alkB-alaD-TA]. By contrast the oxygen and carbon transfer rates decrease in the case of the deletion mutant W3110 ΔFadE [alkB-alaD-TA] and approach the level observed prior to the biotransformation (FIG. 3). The amount of product formed by both strains is comparable (FIG. 4), in fact the yield is slightly better in case the mutant is used.

10 minutes after completion of the biotransformation a clear phase separation could be visually detected in the reaction vessel comprising the strain W3110 ΔFadE [alkB-alaD-TA], wherein the upper phase comprised approximately 40% and the bottom phase comprised approximately 60% of the volume. A thin inter phase could be observed between the phases. Samples were taken from the upper and lower phase, transferred into a 15 mL falcon tube and spun down at 5500×g for 10 minutes. The tube comprising the sample from the lower phase comprised approximately 95% aqueous phase and biomass. The tube comprising the sample from the upper phase comprised approximately 60% organic solution (FIG. 2). The reaction vessel comprising strain W3110 [alkB-alaD-TA] contained a homogenous emulsion after 10 minutes, and no phase separation could be observed for another 20 minutes (FIG. 1).

In summary, deletion of the gene encoding FadE, the E. coli acyl-CoA dehydrogenase of the β-oxidation pathway, leads to an improved phase separation if the mutant is in an aqueous solution and contacted with a water-immiscible organic solvent as well as to a lower consumption of oxygen. 

1. A microorganism having a reduced fatty acid degradation capacity and expressing a recombinant alkane oxidase.
 2. The microorganism according to claim 1, wherein the expressed recombinant alkane oxidase is selected from the group consisting of a rubredoxin-dependent alkane oxidase, a cytochrome P450 enzyme, a xylene monooxygenase, a methane monooxygenase, and variants thereof.
 3. The microorganism according to claim 1, wherein the fatty acid degradation capacity is reduced due to deletion of a gene encoding an enzyme selected from the group consisting of a fatty acid importer, a fatty acid-CoA ligase, an acyl-CoA dehydrogenase, a 2,4-dienoyl-CoA reductase, an enoyl-CoA hydratase and a 3-ketoacyl-CoA thiolase.
 4. The microorganism according to claim 1, wherein the microorganism is a prokaryotic or a lower eukaryotic cell.
 5. The microorganism according to claim 1, wherein the microorganism expresses a recombinant alcohol dehydrogenase.
 6. The microorganism according to claim 1, wherein the microorganism expresses a recombinant transaminase.
 7. The microorganism according to claim 6, wherein the microorganism expresses a recombinant amino acid dehydrogenase.
 8. A method for oxidizing an alkyl, comprising a) contacting the alkyl with an aqueous solution comprising the microorganism according to claim
 1. 9. The method according to claim 8, further comprising b) contacting the aqueous solution from a) with a water-immiscible organic solvent.
 10. The method according to claim 9, wherein b) is carried out following completion of the alkyl oxidation.
 11. (canceled)
 12. The method according to claim 8, wherein the alkyl is a compound represented by the formula H—(CH₂)_(x)—R, wherein x is 1 to 30, and R is any chemical group.
 13. The method according to claim 8, wherein the alkyl is a linear alkane.
 14. The method according to claim 9, wherein the water-immiscible organic solvent is a water-immiscible fatty acid or fatty acid ester.
 15. The microorganism according to claim 1, wherein the microorganism is a bacterial cell.
 16. The microorganism according to claim 1, wherein the microorganism is E. coli.
 17. The microorganism according to claim 7, wherein the recombinant amino acid dehydrogenase is an alanine dehydrogenase. 